To reach a better understanding of the spatial variability of water quality in the Lower Mekong Basin (LMB), the Self-Organizing Map (SOM) was used to classify 117 monitoring sites and hotspots of pollution within the basin identified according to water quality indicators and US-EPA guidelines. Four different clusters were identified based on their similar physicochemical characteristics. The majority of sites in upper (Laos and Thailand) and middle part (Cambodia) of the basin were grouped in two clusters, considered as good quality water with high DO and low nutrient levels. The other two clusters were mostly composed of sites in Mekong delta (Vietnam) and few sites in upstream tributaries (i.e., northwestern Thailand, Tonle Sap Lake, and swamps close to Vientiane), known for moderate to poor quality of water and characterized by high nutrient and dissolved solid levels. Overall, we found that the water in the mainstream was less polluted than its tributaries; eutrophication and salinity could be key factors affecting water quality in LMB. Moreover, the seasonal variation of water quality seemed to be less marked than spatial variation occurring along the longitudinal gradient of Mekong River. Significant degradations were mainly associated with human disturbance and particularly apparent in sites distributed along the man-made canals in Vietnam delta where population growth and agricultural development are intensive.